Synopsis of nonenveloped DNA viruses and entry and exit of endosomes
Endocytosis is critical for the internalization and maturation of many DNA viruses. Since all non-enveloped DNA viruses replicate in the nucleus, they can also take advantage of endocytosis trafficking for transport from the cell surface to the nucleus. The internalization often involves clathrin-mediated endocytosis (ClaME), but also macropinycytosis, caveolae-mediated endocytosis (CavME) and other less characterized internalization mechanisms. Viruses strive to avoid evidence of cell entry, and thus of immune attack, and therefore as much as possible take advantage of existing systems. Consequently, virus entry has been extensively studied to understand endocytosis. In contrast to inert cargo, like dextran, well-defined virus particles can be mutated to change biological properties, e.g. receptor interaction, maturation steps or penetration of the endosomal membrane to the cytosol, and thus provide additional tools for the study of endocytosis. However, the use of viruses also poses major challenges since (i) the virus particle population may be heterogeneous; (ii), only one out of a thousand particles may be infectious; (iii), viruses may be using different pathways at the same time; (iv), viral entry pathways may depend on the cell types used; (v), some viruses, eg. papillomaviruses are difficult to reproduce in tissue culture; (vi) the mobility of the infecting particles may be heterogeneous; and (vii), viruses often developed mechanisms to avoid infected cells (e.g. viral neuraminidases). The use of purified viruses and cell cultures, although imparting useful models, may not reproduce the
2. Overview of non-enveloped DNA viruses and tools to study their entry
2.1. Non-enveloped DNA viruses
Viruses can be used as a tool to study endocytosis since their defined structure can be modified specifically by site-directed mutagenesis to study different parts of the endocytic pathways. Functions related to virus entry are embedded in the virus capsid. Therefore, we will summarize here the structures and properties of these viruses. All, except the iridoviruses, have been solved by X-ray crystallography and their properties are recapitulated in Table 1.
Circoviruses are among the smallest DNA viruses; the diameter of the icosahedral particle is about 20 nm. The virus has a circular, 2 kb single-stranded DNA genome (ssDNA). The T=1 capsid consists of 60 subunits (1, 2). The best-known species infect pigs and birds. Soluble monomers of the porcine circovirus 2 (PCV2) capsid have been expressed in
The icosahedral viral capsid of parvoviruses infecting vertebrates is made up of two or three proteins that have large common C-terminals part and different N-terminal extensions, due to different translation initiations or proteolysis. The rugged capsid structure is very resistant to acids, bases, solvents such as chloroform or butanol, and temperatures to beyond 50 C. The X-ray structures of many parvoviruses have been solved, but these structures lack the N-terminal extensions (5-10). Parvoviruses, such as the minute virus of mice (MVM), package their linear ssDNA genome into preformed capsids, in a process that is probably driven by a virus-encoded NS1 (helicase). Each of the twelve 5-fold vertices have a roughly cylindrically shaped pore created by the juxtaposition of 10 antiparallel beta-strands, two from each of the 5-fold-related capsid proteins. Mutant capsids that have their 5-fold channels blocked are unable to package DNA, strongly suggesting that the 5-fold pore is the packaging portal for genome entry (11). It may also be the DNA delivery site after the conformational changes occurring in these pores in the endosome. Virtually all parvoviruses also contain a phospholipase A2 activity (PLA2) in the N-terminal extension of the largest capsid protein (12, 13). This domain is located inside the capsid and needs to be externalized for it to be able to breach the endosomal membrane barrier (12). These multistep conformational changes occur in the endosomes.
|Family||Structure2||Capsid composition||Entry mode||Escape|
|PDB : 3R0R||T=1|
|ClaME||Uncoating by serine proteases;|
Escape early (not at very low pH)
|PDB : 2CAS||T=1|
|LE / lysosomes|
Viral PLA2 activity
|PDB : 1SVA||T=7|
360 VP1 proteins
|CavME||ER, Viral VP2 cellular proteins BAP31 and BiP|
|PDB : 1L0T||T=7|
360 L1 proteins
viral capsid protein L2
|PDB : 1VSZ||Pseudo-T=25|
720 hexon proteins
12 penton bases
Penton base, L3/p23 protease, protein VI.
|Iridoviridae||PDBe3 : 1580||T=147|
1460 hexon proteins
(each forms trimer)
Fiber proteins (CIV)
|May involve partialuncoating and internal membrane|
The 45 nm diameter T=7 icosahedral particles polyomaviruses virions (SV40, BK virus) are non-enveloped. The crystal structure of SV40 has been solved and found to be organized unlike other viruses (14-16). The 3 capsid proteins, VP1-3, form 72 pentameric capsomers, 60 hexagonally coordinated plus 12 pentamerically coordinated (at the vertices), i.e. the capsomers at the 5-fold axis have all 5 neighboring capsomers whereas the other 60 have 6 capsomer neighbors via different intercapsomer interactions. The 72 pentameric capsomers in each virus particle contain together 360 copies of VP1 plus 30-60 copies each of VP2 and VP3, i.e. ~1 copy in the tapering cavity inside of each pentamer. VP2 is important during cell entry. Each copy of VP1 has a sialic acid binding site on the surface and these are part of the receptor-binding site for the virus; hence the particles have haemagglutinating properties. The long C-terminal arms of VP1 stabilize the interpentameric contacts by invading neighboring capsomers. VP2 is myristylated at its NH2-terminus, and is also believed to be important in holding the particle together (17). Recently, it was also shown that the cysteine at position 9 in VP1 forms C9-C9 disulfide bonds and thus contributes to intercapsomeric crosslinks (18).
Previously, the Papovaviridae family contained both the Papillomaviridae and the Polyomaviridae. Now, these two are separate families but their earlier co-classification indicates a certain degree of similarity (19, 20). The diameter of the papillomaviruses is about 20% larger than that of polyomaviruses, mainly due to the larger L1 as compared to VP1 (21, 22).The atomic structure resembles that of polyomaviruses (23), in particular the core capsomers, and the 55 nm diameter T=7 icosahedral particles also have 72 pentameric capsomers (20, 24, 25). However, the cysteine residues involved in intercapsomer disulfide bonds are C175 and C428 (23).
The over 50 serotypes of the
Iridoviruses have recently been reviewed in detail (29). Currently, the
2.2. Tools used to study endocytosis and trafficking of viruses
Transmission electron microscopy (TEM) is still widely used, and enables visualization of virus in endocytic cups (size and shape, presence of coats). For instance, TEM imaging demonstrated that Adeno-associated virus 5 (AAV5) can enter cells in non-coated vesicles, pointing to a non-clathrin mechanism (32). Immunohistochemistry using gold-labeled antibody can be used in TEM to colocalize structures. Scanning electron microscopy (SEM) facilitates 3D imaging that can be helpful to see ruffles on cell surface, as shown in an AAV2 study (33).
Fluorescence, particularly confocal microscopy, is an excellent tool to visualize viruses and cellular structures. It is frequently used to confirm use of a pathway by co-localisation of the virus and pathway components. It can also be used in live imaging to see the movements of the virus along cellular structures, such as plasma membrane (filopodia) and actin or tubulin structural networks. One should be aware that conformational epitopes may change during the endosomal pathway and that some viruses have very high GCE/FFU ratios (genome copy equivalent/foci forming unit), so that an excess of non-infectious virus in the viral preparation could mask the real infectious pathway. For further details, see chapter by Aaron and Timlin.
2.2.2. Chemical inhibitors
Chemical inhibitors are widely used and provide an excellent basis for endocytosis studies of a particular cargo (34) (Table 2). These are usually not expensive, easy to work with, and have the major advantage of providing a uniform treatment of the cells. None of the extensively used pharmacological inhibitors have complete specificity, but, some parameters can be controlled to limit the possibility of artefacts. Dose-response curves to establish lowest concentrations and shortest exposition times possible to limit side effects on the cells are valuable, and limit upregulation of compensatory mechanisms. It is also easy to wash out the inhibitors to recover a normal state of the cells, in the case of reversible inhibition. This also remove virus that did not yet penetrate the cells, and allows continuing the infection with only endocytosed virus in normal conditions of the cells. Confirmation with another inhibitor, without the same side effects, is convenient. A combination of methods with siRNA or dominant negative/constitutive mutants can be exploited to identify the entry pathway of a specific cargo. Readers are referred to selected literature for further details on any particular inhibitor for comprehensive discussions (34, 35).
2.2.3. Dominant-negative/constitutive mutants
Dominant-negative mutants drive the expression of non-functional versions of the protein of interest (Table 3). The regular protein is still present but strong expression of the mutant form will result in an inactive pathway (35). The main challenges using dominant-negative mutants is to choose the appropriate protein and to achieve high efficiencies in the transfection of the constructs. Stable transfection, with selection of the transfected cells, is not a good choice, since long (constitutive) treatments that donwnregulate a pathway is prone to result in the upregulation of compensatory pathways that could be used by the cargo. Using dominant-negative mutants can also be more time-consuming and become more costly than chemical inhibitors. A constitutively active protein can block a pathway at a certain step, e.g. constitutively active Rab5 protein inhibits the passage from early endosomes and late endosomes (see section 4.2.2.). There is also a complex signalisation process involved in endocytosis, and any modification can have some side effect on the cells.
|Pathway||Inhibitor||Mechanism of action||Side effect|
|Clathrin||Hypertonic sucrose||Dispersion of clathrin sub-units, preventing basket formation||Inhibits CavME and macropinocytosis (high doses);Induces important remodelling of the actin network|
|Chlorpromazine||Clathrin and AP2 relocalized at the endosomes||Inhibits phagocytosis and formation of large vesicles|
|Potassium depletion||Prevents clathrin basket formation||Reduces other non-clathrin endocytosis mechanisms; Alteration of actin cytoskeleton|
|Cytosol acidification||Blocks budding of vesicles||Inhibition of macropinocytosis;|
Alters actin cytoskeleton
|Caveolae||Nystatin||Induces cholesterol sequestration and aggregation||Can affect macropinocytosis at high concentrations|
|Methyl-β- cyclodextrin||Disperse cholesterol, mislocalisation of caveolin-1||High concentration leads to inhibition of ClaME and fluid phase endocysotis;|
Affects actin structure
|Cholesterol oxydase||Relocalization of the caveolin-1 to the Golgi||Action of the enzyme is sensitive to several factors that may lead to false negatives|
|Macropino-cytosis||Amiloride||Inhibits Na+/H+-ATPase exchangers;|
Prevents membrane extension formation
|Can affect ClaME;|
Can reduce lipid raft internalization;
Alters actin structure
|Cytochalasin D||Prevents membrane projection by actin||Other mechanisms depend on actin and membrane modelling|
|Wortmannin||Inhibition of PI3K, important for formation and fusion of vesicles||Not exclusive for macropinocytosis|
|Acidification of endosomes||Bafilomycin A||Block the V-ATPase proton pumps||No effect on recycling pathway, prevents transfer from EE to LE|
|Weak base (NH4Cl, chloroquine)||Gently raise the pH of the compartment;||Does not affect the transport of cargo to the lysosomes|
|Pathway / component||Protein||Mode of action|
|ClaME||EPS15||Deletion of EH domains results in loss of recruitment of clathrin and AP2 at endocytosis site (36)|
|Clathrin||Overexpression of the HUB domain in the cells leads to the inhibition of the cage assembly at endocytosis site.|
|CavME||Caveolin-1||Overexpression of the WT protein stabilizes the caveolin structure located at the plasma membrane, thus prevent membrane curvature and/or vesicle budding|
|Caveolin-3||Deletion of N-terminal, or fusion with EGFP in N-terminal inhibits endocytosis (37)|
|Dynamin||Dynamin-1/ Dynamin-2||K44A is widely used. Dynamin is involved in ClaME, CavME and other less described non-ClaME/ non-CavME mechanisms|
|Macropino-cytosis||Pak-1||Important in the regulation of vesicle cycling|
|Rac-1||Important for membrane ruffling, inhibited with N17Rac1(33); Blocks cell cycle in G2/M phase|
|Other pathways||GRAF1||Key player in the newly described CLIC/GEEC pathway (33, 38)|
2.2.4. Gene silencing
Another way to target a particular protein, as opposed to chemical inhibitors and dominant-negative mutants, is gene silencing. Several endocytosis studies use transfection of small interfering RNA (siRNA) or small hairpin RNA (shRNA) to specifically knock down key proteins of different endocytosis pathways (see section 4.3). This method is relatively simple and selective, but depends on good delivery of the interfering RNA. A good knockdown of the target protein needs to be confirmed by Western blot analysis, and only then can endocytosis studies can be performed. However, since there are some entry pathways not fully described yet, it remains a possibility that some proteins that we believe specific to a certain entry mode will be demonstrated in the future to be involved with other pathways.
3. Docking of virus to cell surface and induced signalling pathways
3.1. Virus attachment to cells
The first interaction between a virus and a cell is crucial to promote endocytosis. In its simplest form, the virus binds to a cell surface receptor and triggers directly its endocytosis. However, several viruses have either multiple receptors, or they also bind abundant structure on the cell surface, e.g. as sialic acids, before binding to the “real” receptor. In its most sophisticated form, the virus needs to bind to a primary receptor to trigger conformational changes that will allow binding to a secondary receptor that will in turn trigger endocytosis. Binding to the cell surface is also the first element that can determine viral tropism, if the virus uses a specific receptor present only on certain cell type.
3.1.1. Cell attachment by circoviruses
Monocyte/macrophage lineage cells are target cells PCV2 replication. Misinzo et al. (4) used the porcine monocytic cell line 3D4/31, that supports PCV2 replication in vitro, and glycosaminoglycans (GAG), used by several viruses as receptors, for attachment studies. They observed that heparin, heparan sulfate (HS), chondroitin sulfate B (CS-B), but not CS-A, and keratan sulfate reduced PCV2 infection when these GAG were incubated with PCV2 prior to and during inoculation of 3D4/31 cells. Also, enzymatic removal of HS and CS-B prior to PCV2 inoculation of 3D4/31 cells significantly reduced PCV2 infection. Similarly, when PCV2 virus-like particles (VLP) were allowed to bind onto 3D4/31 cells in the presence of heparin and CS-B, attachment was strongly reduced. This was confirmed for the wild-type virus. Together, these results demonstrated that HS and CS-B are components of the attachment receptors for PCV2.
3.1.2. Cell attachment by parvoviruses
3.1.3. Cell attachment by polyomaviruses
SV40 binds cell surfaces via major histocompatibility complex 1 (MHC-1) (37). This binding provokes the clustering of the virus in lipid rafts, and entry by CavME. Infection can be efficiently inhibited by pre-incubation of the cells with antibodies directed at the MHC-1. It was observed that initial binding (when endocytosis is inhibited by incubating the cells at 4 C) occurs outside of the lipid raft, and that at that point, there was no co-localisation with caveolin (37). However, when the cells are at 37 C, translocation to the caveolin-positive domain is fast and efficient.
3.1.4. Cell attachment by papillomaviruses
Uptake of papillomaviruses is very slow, and the virus can stay at the cell surface for several hours and the half-time of uptake can be as high as 14 hours. A major issue with this family of viruses that have slowed progress in their research was that, in the host, viruses only complete their replication cycle in terminally differentiated keratinocytes (44). Thus, it was only since the 2000’s that virus could be efficiently produced, using “packaging cell lines” (45, 46). It is also hard to infect cells
3.1.5. Cell attachment by adenovirus
The first high-affinity interaction between the cell and the virus occurs between the globular knob of the C-terminal segment of the viral fiber protein and the cellular CAR (Coxackie and Adenovirus Receptor, belonging to the Ig superfamily) or CD46, for sub group C and B adenovirus respectively. The second interaction, of low affinity is between the RGD motive on the viral penton base and the cellular integrins. Together they will form a strong and irreversible connection between the cell and the virus, leading to endocytosis (48).
3.1.6. Cell attachment by iridovirus
Little is known about iridovirus receptors; although the central fibers of the capsomers have been proposed to be involved (31) not all iridoviruses have these fibers. Eaton et al. showed that anti-FV3 serum present at the time of FV3 (
3.2. Induced signalling pathways
Signalization from cell surface is an important process in coordination of different events of an organism. Readers are referred to two extensive reviews on the subject for further details (50, 51). Signalling also regulate endocytosis processes. Binding to a receptor can trigger signalling pathways that promote endocytosis by upregulating the quantity of clathrin at the cell surface. Src kinase was shown to increase new clathrin basket formation by phosphorylation of the clathrin, leading to assembly of the triskelions (52). Activation of p38 increases Rab5 recruitment to plasma membrane, promoting endocytosis (53). Raf kinase is implicated in recycling of the transferrin receptor to the cell surface (54). In the case of viruses, binding of the virus to the cell surface receptor can trigger other endocytosis mechanisms such as macropinocytosis which is not a constitutive process (55). Differential endocytosis can also be used by the cell to control the level of signalling. For example, activated EGFR can be endocytosed by ClaME or CavME. CavME is used when the level of activation is particularly high and lead to degradation of the receptor, while ClaME leads to recycling of the receptor.
4. Entry mechamisms
4.1. Initial steps of entry
Viruses have developed multiple ways to hijack cellular entry processes ideally without any remaining trace at the cell surface to prevent detection by the immune system. The three best understood mechanisms are clathrin-mediated endocytosis (ClaME), caveolae endocytosis (CavME) and macropinocytosis (Figure 1).
ClaME is probably the best characterized cellular uptake mechanism. Cellular uptake was first observed using transmission electron microscopy (TEM) in the 1960’s. One main characteristic is the assembly of clathrin proteins forming basket shape vesicles. As a result it was postulated that the size of material that can be taken via ClaME is limited in order to form the basket clathrin cage. ClaME proceeds in five steps (56). First, it requires specific receptor engagement to trigger intracellular signaling required for the beginning of membrane modeling, a process called “nucleation”. The key element is the highly conserved adaptor protein AP2, recruited from the cytoplasm to the membrane. The next step is the “cargo selection”. At this stage there will be several interactions between the cargo, its receptor and several adaptor and accessory proteins. The tetrameric AP2 adaptor protein, like clathrin, has disproportionally large numbers of interactors, such as dynamin and Eps15, and is a hub of protein networks. Accessory proteins may swap from the AP2 appendage hub to the clathrin hub during coated pit assembly. Clathrin protein will also be recruited at the membrane in the “clathrin coat assembly” step. Clathrin proteins are recruited as triskelions and will be polymerized into the basket shape forming pentagons and hexagons. When the polymerization reaches the point where only a neck shape remains between the forming coated vesicle and the plasma membrane the dynamin protein will be recruited to perform the “vesicle scission” step required for the budding. GTP hydrolysis will be necessary to complete the process. The final step will be the “uncoating and recycling” step. This uncoating is important before the nascent vesicle can move on and fuse with the endosomes. After this, there will be a sorting step deciding whether the vesicle is simply returned to the plasma membrane or pushed forward into the endosomal pathway.
CavME main characteristic is its origin in lipid rafts, small domains rich in sphingolipids and cholesterol (57). This endocytosis mechanism thus depends on cholesterol homeostasis of the cell. Caveolin is a membrane protein that binds to the cholesterol, as a result depletion of cholesterol leads to mis-localization of caveolin and inhibition of the pathway. Several components are involved such as caveolin-1 (caveolin-2 can be present but not required, caveolin-3 is present primarily in muscles), dynamin and actin (implicated in caveolin rigidity/motility). Vesicles acquire caveolin coats while forming; the released caveosomes will keep a neutral pH and can interact with the endosomal pathway, the Golgi system, or as in the case of SV40 endocytosis, transit to the smooth endoplasmic reticulum (37).
Macropinocytosis is an active uptake pathway that requires activation in most cells types (58). Activation of the pathway will lead to intense actin and microfilaments modulation and membrane ruffling. By definition pinocytosis is the uptake of fluids and membranes by the cell (“fluid phase endocytosis”). The main difference with other endocytosis mechanisms is that, once activated, uptake of material will not depend on receptor binding. In macropinocytosis, the membrane ruffles will or will not be closed and the size of the macropinosome is not directed by the cargo. The diameter will usually range between 0.5 to 10 µm, and thus is larger than in other endocytosis mechanisms. In contrast to phagocytosis, that is limited to few cell types, most cells are capable of macropinocytosis. The closure of the macropinosomes will require several cellular factors. Myosins will provide contractile activity, and fusion factors together with kinases, GTPases, will help the process. Once in the cytoplasm, macropinosomes will acidify and interact with the endosomal pathway.
Phagocytosis is a pathway limited to specialised immune cells such as macrophages, dendritic cells, neutrophils and monocytes. This pathway is mostly used by bacteria and fungi. Recent studies reported the use of this pathway also by herpes simplex virus (59) and mimivirus (60), both displaying membranes. The non-enveloped DNA viruses that fall in the scope of this chapter do not use it.
Novel pathways were revealed by investigating entry mechanism of viruses. They are often referred to as clathrin independent endocytosis. Since molecular components involved in these mechanisms depend not only on cargo but also on cell type, several groups suggested classifying them as dependent or independent of such components. For example, no matter which mechanism is involved the forming vesicle will need to be pinched of the cellular membrane. While ClaME, uses dynamin proteins some clathrin-independent endocytosis, were reported to be dependent on dynamin and some are independent.
4.2. Endosomal pathway
Regardless of the endocytosis pathways, virtually all the vesicles formed during entry will fuse or at least interact with the early endosomes, and acquire the Rab5 and EEA1 markers (61). After endocytosis, cargos enter the highly-coordinated endosomal pathways (62). General endocytosis is a highly active and dynamic process. In a mammalian cell up to 180%, of the cell surface will be endocytosed each hour (62). A simple model of the endosomal pathway is shown in Figure 2.
The first compartment encountered is the early endosome (EE) which is slightly acid (pH~6.5), highly pleomorphic, large and tubular shaped. The EEs are the main organelle where sorting will take place. The main markers of EEs are Rab5 and EEA1 proteins. The tubular projection contains microdomains for sorting, and will generate vesicles targeted to the recycling endosomes (with Rab4 and Rab11), or the
LEs have an increased regular oval shape, their pH is more acidic (pH~5.5), and they contain many internal vesicles (62). LEs are generated close to the cell surface and will mature while moving towards the nuclear periphery. Fusion events will occur between LEs, and between LE and lysosomes. The lysosome is highly acid (pH<5) and contains various hydrolases. Acidification of the endosomes is provided by proton pumps within the membrane of the endosomes, the V-ATPases. The gradual acidification of the pathway is regulated by the concentration of these pumps, the isoforms present, and the presence of other complexes. Markers of the lysosomes are the LAMP proteins. In addition to transport of cargo to the lysosomes, there is also transport of cellular components to the lysosomes. The different hydrolases and membrane proteins are renewed to conserve the lysosome integrity, acidic pH and activities. Transport comes mostly from the TGN via the late endosomes. Mobility of the endosomes is dynamic, and involves both major transport on actin and the microtubule (Mt) networks. Also, transport is not unidirectional, but oscillating, since the vesicles interact both with the kinesins (transport toward cell periphery) and the dyneins (transport toward nucleus) on the Mts. As the maturation of the endosomes proceeds, the net movement will be toward the nucleus (64). Movement of the LEs toward the nucleus is important to reach the lysosomes (mostly located in the nuclear periphery). The actin network is also important for transport of the EEs to the LEs, and to generate small vesicles from the EEs that are targeted to the TGN or from LEs toward lysosomes. Actin is also important for fusion between the LEs and lysosomes.
There are more than 60 different Rab GTPases that have specific membrane localizations. Rab small GTPases are important regulators of the endosomal pathway (65) and thus they can be used as markers, or as inhibitors of specific parts of the pathway by expression of dominant-negative form. The regulation switch is the association of the Rab protein with either GDP or GTP, (66). Rab proteins are involved in multiple events. Rab5 is implicated in the recruitment of the clathrin subunits, with other regulator proteins, such as GAPGDI, will enable uncoating of the vesicle. Rab proteins were also shown to interact with actin (myosin) and microtubule (dynein and kinesin) motors. An example is the Rab11 family of proteins that link recycling vesicles to myosin (67). Also an effector of the Rab7 protein interacts with the dynein motor to promote minus-end traffic on the microtubules, transporting late ensosomes toward the lysosomes. Membrane fusion also involves several proteins, including the Rab protein, and SNAREs (soluble NSF attachment protein receptor) (68). In this case Rab protein could mediate targeting of the appropriate membrane and docking of the membranes, for SNAREs to achieve fusion event.
4.3. Pathways selected by various non-enveloped DNA viruses
Members of the same family or the same genus may use different pathways. There is growing evidence that certain viruses evolved the use of multiple entry pathways. This can be an advantage to infect different cell types. However, it can be difficult to determine whether multiple entry pathways are involved or if one complex pathway use different components. Often viruses can have multiple sequential binding to the cell surface, which can trigger different signaling pathways inside the cell, calling for components normally observed to drive different entry pathways.
4.3.1. Endocytosis of circoviruses
Porcine circovirus (PCV) infects a wide variety of cell types, including hepatocytes, cardiomyocytes, and macrophages via an unknown receptor. PCV utilizes ClaME to enter the cell, though other pathways may be involved (69, 70). In contrast, although a dynamin- and cholesterol-independent, but actin- and small GTPase-dependent pathway, allows PCV2 internalization in epithelial cells that leads to infection, a clathrin-mediated PCV2 internalization in epithelial cells is not followed by a full replication (69). Recent evidence suggests dendritic cells (DC) are involved through their particularly elevated endocytosis of the virus. PCV2 can accumulate to high levels both
4.3.2. Endocytosis of parvoviruses
Adeno-associated viruses (AAVs) are members of the
CLIC-GEEC (CLathrin-Independent Carrier/GPI-anchored protein-Enriched Endosomal Compartment) is a clathrin/caveolin independent entry pathway, identified in the past 10 years Figure 3. This pathway is constitutive, and thus attractive for pathogens. The size and shape of the vesicles can vary greatly, and could potentially accommodate large or multiple viruses. An important protein is GRAF1, serving as a marker or by inhibiting the pathway by gene silencing. Another interesting feature of the pathway, for viruses, is that resulting vesicles will acidify, and can provide proper environment for conformational changes.
Adeno-associated viruses type 2 (AAV2) endocytosis was recently extensively studied, and revealed a complex mechanism (33). This endocytosis was independent of clathrin and caveolin. Also, only one known inhibitor (EIPA) of macropinocytosis could affect the entry of the virus. This inhibitor was shown to inhibit Rac1 and Cdc42 signaling, thus affecting other mechanisms as well (74). This endocytosis required actin cytoskeleton and cholesterol. Membrane and actin remodeling was observed by TEM and immunofluorescence, respectively. Virus bound to the cell surface was located in lipid rafts. These features led to the recently described CLIC/GEEC pathway (38). Dominant negative mutants revealed the critical role of Arf1 and Cdc42 for AAV2 endocytosis (33). GRAF1 (GTPase Regulator Associated with Focal adhesion kinase) was also demonstrated to be essential in CLIC-GEEC endocytosis, and postulated to act as a “coat” for that pathway (38). Expression of a truncated mutant of the protein, as well as the use of siRNA, both reduced the endocytosis of AAV2 (33). GRAF-1 is not implicated in ClaME or CavME, and thus appears specific to CLIC-GEEC endocytosis. Combination of GRAF1 and dynamin inhibition further diminished entry of AAV2, confirming that both proteins are important for endocytosis of AAV2. Interestingly, endosomes of the CLIC-GEEC pathway are known to acidify quickly and inhibition of acidification reduced transduction by AAVs (33). Then the virus is transported to the Golgi apparatus, and no co-localisation is observed with endosomal markers (Rab proteins).
4.3.3. Endocytosis of polyomaviruses
SV40 is probably the best described virus using CavME (75). Initial observations were made by electron microscopy (TEM) where the virus was not present in a coated vesicle, as opposed to ClaME. Entry is dependent of cholesterol, actin polymerization, dynamin and caveolin (37, 76). SV40 provides a good example of a particular traffic that does not involve classical endosomal pathway (37). Virus can be a great tool when used as a tracer for the endocytosis pathway, by using fluorescent viruses and either regular confocal microscopy or live imaging. Pelkmans et al. showed that SV40 traffic to the ER in two distinct steps (37). They saw, using live imaging that the virus once bound to its receptor will move on the surface of the cell until it reach the caveolae. Then the virus is endocytosed into very small vesicles. These will transport the virus to larger vesicle already present in the cytoplasm. These vesicle contained several virus particle and were caveolin-positive (“caveosomes”). Then the virus left the caveosomes in tubular-shaped small vesicles that moved along the microtubules toward the nucleus. The virus was then delivered to the smooth ER. No co-localisation with clathrin was observed, and inhibition of ClaME does not affect SV40 infection. SV40 was also reported to infect cells that do not express caveolin (77). In these cells, entry involved tyrosine kinases and cholesterol but again was not dependent of clathrin or dynamin (77). Entry could not be reduced when inhibiting ClaME or macropinocytosis. It was also faster than regular CavME, but led to the ER in a microtubule dependent manner similar to CavME. This is a good example of virus adaptation, enabling infection in a wide range of cells. In caveolae-expressing cells, both pathways are used. They share important characteristics such as dependence of cholesterol, tyrosine kinases and transit in neutral vesicles using the microtubule network leading to the ER (77).
BK virus is a polyomavirus that poses serious health problems (78). It causes diseases in immunosuppressed patients after renal transplants. Immunosuppression therapy can enable usually non-threatening viruses to replicate easily leading to diseases. More than 80% of the population has been infected often at young age and latent infection of the kidney is present in 50% of the population. Efforts are being made to understand infection by this virus. It was demonstrated that it hijacks CavME, as its relative virus SV40. Cholesterol depletion efficiently inhibited infection. Knock-down of caveolin-1 by siRNA inhibited the infection, while a knockout of clathrin had no effect. Immunofluorescence and co-localisation with confocal microscopy confirmed that BK virus use CavME in cell culture (78-80).
4.3.4. Endocytosis of papillomaviruses
Papillomaviruses are a good example of different family members using different entry pathways even after binding to similar receptors, thus co-receptors could shunt the virus to different pathways. BPV1 entered cells by ClaME (81), and HPV31 preferred CavME (82). To determine which pathway was important they used chemical inhibitors, dominant-negative mutants and TEM. Another publication showed that for HPV16 and HPV31 macropinocytosis was important in NK cells (83). In this case, membrane ruffling was observed when the cells were incubated with the virus-like particles (VLPs). Then, co-uptake of fluid phase was demonstrated with fluorescent dextran. Inhibition by chemical inhibitors prevented this fluid-phase uptake. In term of RhoGTPases, HPV VLPs induced activation of Cdc42 and inhibition of Rac1. Papillomaviruses are highly asynchronous and their pathways chosen are disputes in the literature.
4.3.5. Endocytosis of adenoviruses
Adenovirus could be observed in clathrin-coated vesicles by TEM (84). It is also present in non-coated vesicles. Dominant-negative constructs confirmed the ClaME (55). The involvement of integrins (85, 86) suggests that adenovirus endocytosis is highly regulated. These integrins activate PI(3)K which in turn induces actin polymerization and promotes adenovirus endocytosis (87). Another observation was that fluid uptake (macropinocytosis) increased with adenovirus infection (55). Amiloride was effective to inhibit infection. This additional infection pathway also required actin, small Rho GTPases and PKC.
4.3.6. Endocytosis of iridoviruses
The enveloped form of the iridovirus FV3 (genus
5. Virus maturation and endosomes escape
Non-enveloped viruses display a variety of strategies to gain access to the cytoplasm of the cell. Conformational changes are required in capsids to expose hydrophobic domains that anchor or disrupt the endosomal membrane during breaching. Parvoviruses have acquired an enzyme for membrane translocation. Virus trafficking is therefore highly temporally and spatially regulated and a plethora of tools have been developed to study these events.
5.1. Endosomal trafficking of circoviruses and escape to cytoplasm
PCV2 enters monocytic cells predominantly by ClaME and requires an acidic environment for infection. After endosomal escape, the virus aggregates in intracytoplasmic inclusion bodies (ICIs). Subsequently, PCV2 closely associates with mitochondria, completing a first cytoplasmic phase and enters the nucleus for replication (91). In epithelial cells, PCV2 is internalized via a clathrin-, caveolae-, and dynamin-independent small GTPase-regulated pathway (69).The latter leads to a more efficient PCV2 replication while the former seems to trap PCV2 leading to accumulation of the virus within epithelial cells. Inhibition of acidification of the endosomal/lysosomal system reduces PCV2 infection of the monocytic cells, indicating the requirement for a pH drop during replication. Surprisingly, inhibiting acidification highly increases PCV2 replication in epithelial cells, indicating that uncoating occurs at another pH. Serine proteases mediate PCV2 uncoating in both epithelial cells and cells of the monocytic cells but may have different optima in these cells (92). It is not clear how this uncoating process impacts on the endosomal escape.
5.2. Endosomal trafficking of parvoviruses and escape to cytoplasm
Parvoviruses have a unique enzyme domain, phospholipase A2, in their capsid (12, 13, 93). This protein domain with this motif is located inside the mature virion, and thus the virus needs to undergo a conformational change in order to expose and use this enzyme. The pentamer at the fivefold symmetry axis has a 0.8 nm central pore and is itself encircled by a 1.5 nm-deep depression (“canyon”). The hairpins of the central cylinder (see section 2.1.2) can thus move outward leading to a greater pore and allowing N-termini of VP2, during the transit in the endosome and acidification (94), to become exposed one by one and their N-termini proteolytically cleaved, yielding successively a larger pore. The internal unique part of VP1 (that resists proteolysis) is then able to externalize and expose its phospholipase A2 domain. Phospholipases catalyze hydrolysis of phospholipids, providing a means to breach the endosomal barrier. This motif is absolutely essential for parvoviruses infection and acts prior to the delivery of the genome to the nucleus (13). Millimolar calcium concentrations present in the endosome but not in the cytosol, are required for optimal activity of the enzyme (12). Since it was required after accumulation of the virus in the perinuclear region, but prior to DNA expression in the nucleus, it was hypothesized that its role could be the ensodomal escape. Co-uptake studies showed that small dextran particles, but not α-sarcin, could be released from the endosomes together with parvovirus escape. These observations suggested that parvovirus escape implicated small pore formation and not broad endosome disruption. Viruses harboring mutations in the PLA2 enzymatic domain remained trapped in endosomes in perinuclear areas, but could be complemented in
5.3. Endosomal trafficking of polyomaviruses and escape to cytoplasm
SV40 enters via caveolin-1 containing vesicles that budded from the caveolae and that are transferred to caveosomes (lack markers for endosomes, lysosomes, ER and Golgi) (37). SV40 is then transferred from caveolin-free, tubular vesicles to the ER via an intermediate COP1 compartment that is brefeldin A-sensitive (95). Interestingly, polyomavirus is delivered to early endosomes and a crosstalk between ClaME and CavME has been reported for JCV (96). Nevertheless, after passage through early endosomes these are sorted to caveosomes. Escape occurs from the ER to the cytosol before reaching the nucleus (97). An elegant and elaborate study in an attempt to describe mechanisms involved in this escape was published in 2011 (97). TEM studies suggested profound conformational changes in the ER where the capsid diameter decreased from 45nm to 34nm. These conformational changes led to exposing structural protein VP2, which was not accessible in the mature virion that entered the cell. This VP2 protein contains hydrophobic structures that interact with the ER membrane. In the absence of VP2, the capsid can be targeted to the ER, but would not be able to transit back to the cytosol. There are several cellular proteins essential for SV40 escape to the cytosol. BAP31, highly-abundant membrane protein of the ER, is essential for SV40 infection. The chaperone BiP is also important for the escape, and will play its role after BAP31-induced conformational changes in the capsid suggesting that the ERAD-factors (isomerases, Derlin) assist is in SV4 ER- cytosol dislocation as used for misfolded proteins (97).
5.4. Endosomal trafficking of papillomaviruses and escape to cytoplasm
This subject is, due to the lack of suitable study models, still controversial. Conformational changes occur quickly after HPV entry and capsid protein L1 conformational epitope becomes inaccessible after endocytosis (44). Uncoating can be observed by L2 detection (normally buried and inaccessible in mature virions), or DNA detection. Papillomavirus escape from the endosomal pathway, at a relatively early stage, and only partial co-localisation between the virus and the lysosome can be seen (44, 98). Structural protein L2 is a major player in the escape. The protein has a hydrophobic C-terminal, adjacent to positively-charged residues, believed to destabilize the membrane structure and leading to disruption. It was recently described that the cellular protein nexin-17 was also important (98). The suggested role of this protein is to retain the virus in the late endosomes, preventing degradation in the lysosomes. When this protein is knocked down, strong co-localisation of the virus with the lysosomes was observed. Importantly, the binding sequence in L2 protein is conserved among
5.5. Endosomal trafficking of adenoviruses and escape to cytoplasm
During entry, fibers are released, the penton base structures dissociated, the proteins connecting the DNA to the inside surface of the capsid degraded or shed, and the capsid-stabilizing minor proteins eliminated (100). The uncoating process starts immediately upon endocytic uptake with the loss of fibers and ends with the uptake of dissociated hexon proteins and DNA into the nucleus. For the adenoviruses, the escape will occur quickly, and is postulated to be as early as the sorting endosomes (48). An acidic pH is not sufficient for conformational changes in the virus structure to occur but a large number of factors, such as integrin, are involved (101). Integrin binding together with CAR-mediated drifts supported fiber shedding from adenovirus particles, leading to exposure of the membrane-lytic internal virion protein VI and enhanced viral escape from endosomes (102). The Ts1 mutant, which lacks the viral protease and cannot process the capsid or release the fibers during endosomal trafficking, will not be able to breach the endosomal membrane and ends up in lysosomes where it is degraded (103).
For subgroup B adenoviruses, Kalin et al. showed by confocal laser scanning microscopy, electron microscopy, and live cell imaging (104) that Ad35 colocalized with fluid-phase markers in large endocytic structures that were positive for CD46, alphanu integrins, and also CtBP1 (89).Their results extended observations with HAdV-3 (Ad3), using chemical inhibitors and dominant-negative mutants, that macropinocytosis is an infectious pathway for subgroup B human adenoviruses in epithelial and hematopoietic cells.
5.6. Endosomal trafficking of irdoviruses and escape to cytoplasm
Maturation of iridoviruses during entry has not been investigated in detail. The minor zip and finger proteins may play an essential role for capsid destabilization. Uncoating may occur at the nuclear membrane but, unlike herpesviruses, iridovirus genomes are not infectious, indicating that virion-associated proteins are required to initiate viral gene transcription (105). Early EM studies showed that FV3 ended up in the lysosome (88); however this may be a non-productive pathway. It has been suggested that ISKNV traffics via the caveosome to the ER, similar to SV40, and TFV via the caveosome to the
Endocytosis is a wide subject that has been studied for a very long time. Viruses are definitely great tool to study endocytosis since they are masters in hijacking every single possibility of the cell to achieve infection. Elucidating steps in viral replication often leads to hitherto unknown mechanisms that expand our knowledge of normal cellular processes. It is also interesting to realise that significant differences are found inside a family of closely related viruses. Co-evolution with different hosts, in different conditions pushed viruses to adapt in different direction and provide a great variety of tools. However, in the case of life threatening or economically-important viruses, it is sometimes very hard to find a way to prevent infection without impeding with normal cellular processes. This growing field of research will surely provide new exiting insights to previously unknown cellular process, and will continue to provide good targets for antiviral drugs.
Authors wish to thank Natural Sciences and Engineering Research Council of Canada for grant to support P.T. and for the scholarship to M.B.